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• What is an Aptamer?
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The SELEX Process

Aptamers are generally discovered using the in vitro selection process referred to as SELEX (Systematic Evolution of Ligands by EXponential enrichment). SELEX is an iterative process used to identify an aptamer to a chosen molecular target from a large pool of nucleic acids. The process relies on standard molecular biological techniques, which allow a single researcher to conduct multiple selection experiments in parallel. SELEX was first developed in the early 1990's by the Gold group at the University of Colorado. Since that time, it has been used to identify high-affinity aptamers against hundreds of targets.

Step One: Pool Generation

At the outset of a SELEX experiment, a pool of nucleic acid molecules is generated using standard automated oligonucleotide synthesis methods. Archemix uses pools comprising a variety of nucleotide compositions for SELEX. Natural RNAs/DNAs have short half-lives in serum due to nuclease degradation. Nuclease activity may be blocked by modifications to the 2'-ribose position (e.g., 2'-O-methyl) on the oligonucleotide backbone. Therefore, in addition to using DNA-based pools, SELEX is also conducted using pools of nucleic acids containing fully 2'-O-methyl nucleotides, or mixtures of 2'-deoxy and 2'-O-methyl nucleotides. At the outset of a selection, each pool contains approximately 10¹⁴ oligonucleotides, each with a unique sequence that can, in principle, adopt a unique three-dimensional structure. A few of these molecules – the aptamers – present a contact surface that is complementary to an "apitope" on the target molecule.

Step Two: Selection

The selection step is designed to find those molecules with the greatest affinity for the target of interest. The library of nucleotide sequences is exposed to the target protein and allowed to incubate for a period of time. The molecules in the library with weak or no affinity for the target have a tendency to remain free in solution, while those with some capacity to bind will tend to associate with the target. Any one of several methods is used to physically isolate the aptamer-target complexes from the unbound molecules in the mixture, and then the unbound molecules are discarded. The target-bound molecules, among which are the highest affinity aptamers, are purified away from the target and used for the subsequent steps in the SELEX process.

Step Three: Amplification

The captured, purified sequences are copied enzymatically, or "amplified", to generate a new library of molecules that is substantially enriched with oligonucleotides that can bind to the target. The enriched library is used to initiate a new cycle of selection, partitioning and amplification. One complete cycle of binding, partitioning and amplification is referred to as a "round" of SELEX.

Step Four: Aptamer Isolation

After five to 15 cycles of the complete SELEX process, the library of molecules is reduced from 10¹⁴ unique sequences to a small number that bind tightly to the target of interest. At this stage, the nucleotide sequences of individual members of the library are determined.  The target binding affinity and specificity of selected sequences are then measured and compared. We advance the aptamers with the highest affinity and functional activity against the target to our post-SELEX modification processes.

Post-SELEX Modification Processes

The aptamers isolated by the SELEX process exhibit affinity and specificity for the selected target, but often exhibit chemical characteristics that may limit their potential as therapeutics. Accordingly, following the SELEX process, we use proprietary chemistry techniques, which we call post-SELEX modification, to design, stabilize and optimize the early lead series of aptamers to create aptamer product candidates for clinical development. Specifically, we seek to engineer the aptamer’s rates of metabolism by and excretion from the body so that the aptamer may have the appropriate duration of action to effectuate the desired therapeutic response.

The steps involved in post-SELEX modification include:

• Minimization. The initial aptamer sequences isolated by SELEX are typically 70 to 80 nucleotides long. Commercializing aptamers of this length would be difficult and expensive using current manufacturing techniques, and production yields would be low. Accordingly, we apply our proprietary methods to identify the active site or core of the aptamer and remove unnecessary nucleotides from the molecule. We are typically able to reduce the aptamer to between 20 and 40 nucleotides in length without compromising the affinity, specificity or functional activity of the aptamer for the target of interest.

• Optimization. Once we have an aptamer of appropriate size, we optimize its affinity, functional activity and metabolic stability.
  
      — Affinity and functional activity improvements. We use sequence and chemical modifications to improve an aptamer’s affinity for its target and functional activity using a technique in which sets of variant aptamers are chemically synthesized. We then compare these variant aptamers to each other and to the starting aptamer in order to determine which modifications improve affinity, functional activity or both.
  
      — Nuclease resistance. If not chemically altered, aptamers composed of unmodified nucleotides may be rapidly degraded, or metabolized, by enzymes which are naturally present in the blood and tissues. These enzymes, known as nucleases, bind to and metabolize the aptamer. While rapid drug clearance and a short duration of action are desirable for some clinical applications, a prolonged duration of action is necessary for other disease categories. Accordingly, we use proprietary methods to identify the specific sites within an aptamer that are most susceptible to nuclease metabolism. With this information, we introduce site-specific stabilizing substitutions into the aptamer to achieve nuclease resistance.

• PEGylation. Duration of action is often correlated to how long the aptamer remains in the body. Because aptamers are small in size, they may be naturally excreted before they have achieved their intended therapeutic effect. To slow the rate of excretion from the body, we increase the size of the aptamer by attaching it to another molecule known as polyethylene glycol, or PEG, to create a larger molecule. This process is known as PEGylation. We can achieve the desired duration of action by using different sizes, structures and attachment locations of PEG molecules. Once we PEGylate the aptamer, we test it to determine whether we have achieved the desired duration of action.

Through this combination of SELEX and post-SELEX modification processes, we are able to design and confirm the desired properties of an aptamer that we believe will address the proposed therapeutic indication.